1. Field of the Invention
This invention relates to an improved process for the fractionation of particles and an apparatus for conducting the same. More particularly, the invention relates to a field-flow fractionation process utilizing a new type of channel construction which permits one to achieve hydrodynamic relaxation and improved sample concentration.
Specifically, the invention provides a new field-flow fractionation process for the separation of samples of particles and macromolecules which uses a modified channel structure to achieve hydrodynamic relaxation without involving a stop-flow procedure and thus provides improved speed of operation as well as improved sample concentration. The new process of the invention comprises an improvement in the field-flow fractionation (FFF) process wherein a sample carrier fluid containing the particles to be separated is forced through a thin flow channel having one or more inlets and one or more outlets and a field or gradient is used to induce a driving force acting across the thin dimension of the channel perpendicular to the flow axis, the improvement of which comprises employing one or more permeable wall sections in the enclosing wall or walls of the channel and forcing substreams of fluid to enter or exit through the permeable wall sections, the said permeable wall sections making up not more than 25% of the total wall area of the channel.
The invention further provides an apparatus for conducting the above-described process comprising an elongated flow channel enclosed by wall elements, means for applying a driving force perpendicular to the long axis of the channel, one or more inlet means for introducing fluid into one end of the enclosed channel, one or more outlet means for withdrawing fluid from the other end of the channel, the enclosing wall elements including one or more permeable wall sections that are so constructed that substreams of fluid can be forced into or withdrawn from the channel through the permeable wall sections, said permeable wall sections making up less than 25% of the total wall area enclosing the channel, and adjustable flow control means for controlling the flowrate of the fluid at the inlet means and independent control means for controlling the flowrate of the fluid being introduced into or withdrawn from the permeable wall sections.
2. Prior Art
There is a growing need in industry and health sciences for the separation and characterization of micron sized particles including biological cells, latices, environmental particles, industrial powders, crystallization products, and related particulate matter. There is also a growing need for the separation of submicron sized particles, macromolecules and synthetic polymers.
Various methods have been proposed, but in general, they have been too slow, complex in operation, inefficient and expensive or have failed to effect the separation with the desired degree of resolution needed for commercial operations.
Some of the highest resolutions techniques disclosed have been those based on field-flow fractionation as disclosed in the following U.S. patents and copending patent applications: U.S. Pat. Nos. 3,449,938, 4,147,621, 4,214,981, 4,250,026, and copending patent application--Giddings--"Lift-Induced Hyperlayer Field-Flow Fractionation Process for Particle Separation" Ser. No. 153,774, filed Feb. 8, 1988, U.S. patent application--Giddings--"High Speed Separation of Ultra-High Molecular Weight Polymers by Hyperlayer Field-Flow Fractionation" Ser. No. 217,707, filed Jul. 11, 1988, and U.S. patent application--Williams--"Process for Programming of Field-Flow Fractionation" Ser. No. 237,188, filed Aug. 29, 1988.
Other methods for particle separation are disclosed in U.S. Pat. No. 4,737,268 and copending patent application Giddings--"Process for Continuous Particle and Polymer Separation in Split-Flow Thin Cells using Flow-Dependent Lift Forces" Ser. No. 194,851, filed May 17, 1988.
Many articles, such as Giddings et al--"Fast Particle Separation by Flow/Steric Field-Flow Fractionation"--Anal. Chem. (1987) 59 1957, have been published disclosing modifications in the particle separation processes.
While the FFF process and modifications as noted above have met with considerable commercial success, the use has been limited for certain operations because of limitations as to speed of operation and loss of sample material due to adhesion to the wall. The basis for such limitations is noted below.
In virtually any kind of field-flow fractionation process, a relaxation step must be carried out in the FFF channel prior to the beginning of effective separation. In the relaxation process, sample material that is distributed widely over the stream lines entering the channel is forced into narrow cross-sectional distributions from which separation is possible. Normally sample is driven close to one wall (the accumulation wall) of the channel during relaxation by the same external field or gradient that is used to implement FFF separation. In most cases the axial flow is halted as relaxation takes place in order to control band distortion and broadening. This so-called stopflow procedure often leads to flow instabilities accompanied by baseline shifts and provides a window of vulnerability in which particles are most susceptible to adhesion to the channel wall. It also increases run time.
Various methods have been proposed to avoid the stopflow procedure. One of the methods 1 have proposed makes use of hydrodynamic relaxation, a process in which the sample is driven close to its equilibrium position by the manipulation of flow rather than by sluggish field-driven transport in the channel. In this method, hydrodynamic relaxation is carried out by using a flow splitter at the inlet end of the FFF channel. Manipulation of the flow rates of the incoming flow streams entering above and below the splitter make it possible to drive the sample, contained exclusively in the substream emerging from below the splitter, close to the accumulation wall of the channel, a position from which separation can quickly commence.
A second flow splitter can be used at the outlet of the channel to concentrate the component materials for enhanced detection. It has been shown that an outlet flow splitter is capable of stripping off the bulk of liquid flowing above the sample layers in the channel, thus leaving the sample in a more concentrated form for detection and collection.
There are, however, several disadvantages with the use of flow splitters in FFF systems for the above purposes. First, for proper operation these splitters must be suspended evenly across the several centimeter wide gap of the thin channel; unevenness amounting to a few tens of micrometers would noticeably distort the hydrodynamic relaxation process. A second difficulty is that the introduction of a flow splitter and the two associated flow spaces on either side of the splitter, three layers in all, is very often inconsistent with the utilization of very thin, say 100-200 .mu.m, high performance FFF channels. Third, since the flow stream in which sample is introduced must traverse the narrow gap on one side of the splitter where the thickness is only a fraction, ususally approximately one third, of that of the full channel, there is an enhanced risk that larger particles in the sample, whether part of the sample or part of an impurity will clog all or part of the streampath needed for sample introduction. Fourth, at high flowrates the abrupt change in flow direction at the splinter edges may introduce eddy currents in the fluid capable of disrupting the distribution of components near the inlet and outlet.
It would therefore be highly advantageous to find a promising method for the modification of the FFF process to achieve hydrodynamic relaxation and sample concentration wihout any of the above-noted disadvantages.
It is an object of the invention, therefore, to provide an improved FFF process which achieves hydrodynamic relaxation without any of the forementioned problems. It is a further object to provide a new FFF process which effects a reduction in the relaxation effect and eliminates the stop-flow procedure. It is a further object to provide a modified FFF process which is capable of effecting separation as a high rate of speed. It is a further object to provide a modified FFF process which gives hydrodynamic relaxation and improved sample concentration. It a further object to provide an improved FFF process which can be adapted to any of the above-noted FFF techniques. These and other objects of the invention will be apparent from the following detailed description thereof.